Thin Layer Identification of Jiedu Shengxue Granules and Determination of Notoginsenoside R1 Content

[Objectives]To establish the quality standard of hospital preparation Jiedu Shengxue granules.[Methods]Scleromitrion diffusum and Prunella vulgaris in Jiedu Shengxue granules were qualitatively identified by thin layer chromatography(TLC).A high performance liquid chromatography(HPLC)was established to determine the content of notoginsenoside R1 in the granule.[Results]The traditional Chinese medicinal materials in Jiedu Shengxue granules could be identified by TLC,and the characteristic spots were stable and clear.Notoginsenoside R1 had a good linear relationship in the range of 10.45-104.5μg/mL,with an average recovery of 98.52%and RSD=2.36%.[Conclusions]TLC and HPLC,as the quality control methods of Jiedu Shengxue granules,have high accuracy and good repeatability,which lays a foundation for the quality control of this mixture.

HPLC测定经络贴巴布剂中3种皂苷的含量

目的建立经络贴巴布剂中三种皂苷成分的含量测定方法。方法采用高效液相色谱法,色谱柱为kromasil-C18(250 mm×4.6 mm,5μm)柱,乙腈-水(梯度洗脱)为流动相,检测波长203 nm,流速1 mL.min-1,柱温40℃。结果人参皂苷Rg1在0.82～8.2μg范围内与峰面积具有良好的线性关系(r=0.999 6),平均回收率为98.33%,RSD=2.723%(n=6);人参皂苷Rb1在0.3256～3.256μg范围内与峰面积具有良好的线性关系(r=0.999 9),平均回收率为99.30%,RSD=0.918%(n=6);三七皂苷R1在0.201～2.01μg范围内与峰面积具有良好的线性关系(r=0.999 8),平均回收率为97.90%,RSD=0.695%(n=6)。结论本方法准确、灵敏、重现性好,阴性无干扰,可用于经络贴中3种皂苷类成分的质量控制。

黄芪甲苷和三七总皂苷中主要有效成分抗PC12细胞氧化损伤的配伍研究

目的探索黄芪甲苷和三七总皂苷中主要有效成分人参皂苷Rg1、人参皂苷Rb1、三七皂苷R1抗CoCl2诱导的PC12细胞氧化损伤的有效配伍剂量。方法采用CoCl2诱导PC12细胞氧化损伤模型,以细胞乳酸脱氢酶(LDH)漏出率为指标,研究4种有效成分对PC12细胞氧化损伤的有效机制浓度。在此基础上按U8*(85)均匀设计实验,确定4种有效成分的有效配伍剂量。结果 4种有效成分都能抑制CoCl2诱导的PC12细胞LDH的漏出,与损伤组相比差异有统计学意义(P<0.05)。且随着药物浓度的增加,对LDH漏出率的抑制作用增强。黄芪甲苷和人参皂苷Rb1在50μmol/L,人参皂苷Rg1和三七皂苷R1在100μmol/L浓度时LDH漏出抑制率约为80%。U8*(85)均匀设计实验多元逐步回归分析得:4种有效成分的8个不同浓度配伍都能抑制CoCl2诱导的PC12细胞LDH的释放,与损伤组相比差异有统计学意义(P<0.05)。人参皂苷Rg1 50μmol/L、黄芪甲苷0.781 25μmol/L、人参皂苷Rb1和三七皂苷R1各1.562 5μmol/L配伍时抗PC12细胞氧化损伤的效应最强。结论人参皂苷Rg1 50μmol/L、黄芪甲苷0.78125μmol/L、人参皂苷Rb1和三七皂苷R1各1.562 5μmol/L配伍组方为抗PC12细胞氧化损伤的有效配伍剂量。

脑得生有效部位三七皂苷类成分含量测定

目的:建立脑得生有效部位中三七皂苷类成分的含量测定方法。方法:采用高效液相色谱法,流动相为乙腈-水,梯度洗脱,流速为0.9 ml/min,检测波长为201nm;柱温为25℃。结果:人参皂苷Rg1的平均加样回收率为99.73%,RSD=1.59%,人参皂苷Rb1的平均加样回收率为99.16%,RSD=1.37%,三七皂苷R1的平均加样回收率为100.34%,RSD=1.90%。结论:该方法操作简便、易行、可用于脑得生有效部位中三七皂苷类成分的含量测定。

Effect of notoginsenoside R1 on hepatic microcirculation disturbance induced by gut ischemia and reperfusion

AIM： To assess the effect of notoginsenoside R1 on hepatic microcirculatory disturbance induced by gut ischemia/reperfusion （I/R） in mice. METHODS： The superior mesenteric artery （SMA） of C57/BL mice was ligated for 15 min to induce gut ischemia followed by 30-rain reperfusion. In another set of experiments, R1 was continuously infused （10 mg/kg per hour） from 10 min before I/R until the end of the investigation to study the influence of R1 on hepatic microcirculatory disturbance induced by gut I/R. Hepatic microcirculation was observed by inverted microscopy, and the vascular diameter, red blood cell （RBC） velocity and sinusoid perfusion were estimated. Leukocyte rolling and adhesion were observed under a laser confocal microscope. Thirty and 60 min after reperfusion, lactate dehydrogenase （LDH）, alanine aminotransferase （ALl＇） and aspartate transaminase （AST） in peripheral blood were determined. The expression of adhesion molecules CD11b/CD18 in neutrophils and tumor necrosis factor- alpha （TNF-α）, interleukin-6 （IL-6） and monocyte chemotactic protein-1 （MCP-1） in plasma were evaluated by flow Oltometry. E-selectin and intercellular adhesion molecule-1 （ICAM-1） in hepatic tissue were examined by immunofluorescence.RESULTS： After gut I/R, the diameters of terminal portal venules and central veins, RBC velocity and the number of perfused sinusoids were decreased, while the leukocyte rolling and adhesion, the expression of E-selectin in hepatic vessels and CD18 in neutrophils, IL-6, MCP-1, LDH, ALT and AST were increased. R1 treatment attenuated these alterations except for IL-6 and MCP-1. CONCLUSION： R1 prevents I/R-induced hepatic microcirculation disturbance and hepatocyte injury, The effect of R1 is related to its inhibition of leukocyte rolling and adhesion by inhibiting the expression of E-selectin in endothelium and CD18 in neutrophils.

Two New Dammarane Glycosides from the Acid Hydrolysis Product of Panax Notoginseng

Two new dammarane glycosides named notoginsenoside T-1 and T-2 were isolated from the mild acid hydrolysis products of the root saponins of Panax notoginseng. On the basis of spectroscopic evidences, their structures were elucidated to be 6-O-beta -D-glucopyranosyl-24(25)-epoxy-3 beta ,6 alpha ,12 beta ,23 xi -tetrahydroxydammar-20(22)(E)-ene 1 and 6-O-beta -D-glucopyranosyl-24(25)-epoxy-23 xi -methoxyl-3 beta ,6 alpha ,12 beta -trihydroxydamm-ar-20(22)(E)-ene 2, respectively.

Role of Notoginsenoside Rg1 in Improving Spatial Cognitive Ability and Lowering Phosphorylation Level of Tau Protein in AD Model Rats

[Objectives] To study the effects and mechanism of notoginsenoside Rg1 on the spatial learning and memory and phosphorylated tau protein in the AD( Alzheimer's Disease) model rat. [Methods]The AD model rat was replicated by injection of Aβ_(25-35) in the left lateral ventricles of SD rats. The low dose( 25 mg/kg),middle dose( 50 mg/kg) and high dose( 100 mg/kg) notoginsenoside Rg1 was used for intragastric administration,respectively,two times every day. After 4 weeks,the Morris water maze test was done to detect the learning and memory capacity,and the immunoblotting,immunohistochemical methods were used to detect the changes in the phosphorylation level and distribution of tau protein in hippocampus of the rats. [Results] After the intracerebroventricular injection of Aβ_(25-35),the learning and memory capacity of the model rats was significantly lower than the learning and memory capacity of the normal control rats. The immunoblotting test results showed that the phosphorylation level of tau protein threonine 231 site( Thr231) in hippocampus was significantly increased,and the nonphosphorylation level was significantly decreased. The morphological testing results showed that the phosphorylation level of tau protein Thr231 of AD model rats was increased markedly in region of DG,CA1 and CA3 of the hippocampus. The intervention of the middle dose notoginsenoside Rg1 could significantly improve the learning and memory capacity of the model rats in Morris water maze. The notoginsenoside Rg1 in three different doses could all reduce the phosphorylation level of tau protein Thr231 in the hippocampal DG,CA1,CA3 regions,and there were no significant differences among the three doses. [Conclusions]The notoginsenoside Rg1 could improve Aβ_(25-35)-induced spatial learning and memory impairment of the AD model rats,and decreased the phosphorylation level of tau protein in hippocampus.

Different Concentrations of Notoginsenoside Rg1 Attenuate Hypoxic and Hypercapnia Pulmonary Hypertension by Reducing the Expression of ERK in Rat PASMCs

Pulmonary arterial hypertension (PAH) is a serious disease which is characterized by increased vascular resistance and pressure. We have previously hypothesized that panax notoginseng saponins (PNS) might attenuate pulmonary vasoconstriction under hypoxia and hypercapnia condition. This study aims to investigate the effect of notoginsenoside Rg1, a main ingredient of PNS, with various concentrations (8, 40, 100 mg/L, respectively) on extracellular signal regulated kinase (ERK1/2) signaling pathway in pulmonary arterial smooth muscle cells (PASMCs). In addition, PASMCs were randomly divided into six groups: SD rat under normoxic condition as control group (N group), hypoxia hypercapnia group (H group), DMSO control group (HD group), Rg1-treatment groups (RgLRgM and RgH group). Western-blot and RT-PCR were used to test the expression of p-ERK protein and the expression of ERK1 mRNA and ERK2 mRNA. This study provided the evidence that the expression of p-ERK protein and the expression of ERK1 mRNA and ERK2 mRNA in HD group and H group were obviously higher than that in N group (P < 0.01), Whereas the level of ERK1/2 mRNA in Rg1-treatment groups was significantly lower than that in HD group and H group (P < 0.01), and the proper concentration of Rg1 is 40 mg/L. These results suggested that notoginsenoside Rg1 can attenuate pulmonary vasoconstriction which may lead to HHPV through reducing the expression of ERK1/2.

Notoginsenoside Ft1 inhibits colorectal cancer growth by increasing CD8^(+)T cell proportion in tumor-bearing mice through the USP9X signaling pathway

The management of colorectal cancer(CRC)poses a significant challenge,necessitating the development of innovative and effective therapeutics.Our research has shown that notoginsenoside Ft1(Ng-Ft1),a small molecule,markedly inhibits subcutaneous tumor formation in CRC and enhances the proportion of CD8^(+)T cells in tumor-bearing mice,thus restraining tumor growth.Investigation into the mechanism revealed that Ng-Ft1 selectively targets the deubiquitination enzyme USP9X,undermining its role in shieldingβ-catenin.This leads to a reduction in the expression of downstream effectors in the Wnt signaling pathway.These findings indicate that Ng-Ft1 could be a promising small-molecule treatment for CRC,working by blocking tumor progression via the Wnt signaling pathway and augmenting CD8^(+)T cell prevalence within the tumor environment.

Pharmacological properties and mechanisms of Notoginsenoside R1 in ischemia-reperfusion injury

Panax notoginseng is an ancient Chinese medicinal plant that has great clinical value in regulating cardiovascular disease in China.As a single component of panax notoginosides,notoginsenoside R1(NGR1)belongs to the panaxatriol group.Many reports have demonstrated that NGR1 exerts multiple pharmacological effects in ischemic stroke,myocardial infarction,acute renal injury,and intestinal injury.Here,we outline the available reports on the pharmacological effects of NGR1 in ischemia-reperfusion(I/R)injury.We also discuss the chemistry,composition and molecular mechanism underlying the anti-I/R injury effects of NGR1.NGR1 had significant effects on reducing cerebral infarct size and neurological deficits in cerebral I/R injury,ameliorating the impaired mitochondrial morphology in myocardial I/R injury,decreasing kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in renal I/R injury and attenuating jejunal mucosal epithelium injury in intestinal I/R injury.The various organ anti-I/R injury effects of NGR1 are mainly through the suppression of oxidative stress,apoptosis,inflammation,endoplasmic reticulum stress and promotion of angiogenesis and neurogenesis.These findings provide a reference basis for future research of NGR1 on I/R injury.

Notoginsenoside Ft1 acts as a TGR5 agonist but FXR antagonist to alleviate high fat diet-induced obesity and insulin resistance in mice

Obesity and its associated complications are highly related to a current public health crisis around the world.A growing body of evidence has indicated that G-protein coupled bile acid(BA) receptor TGR5(also known as Gpbar-1) is a potential drug target to treat obesity and associated metabolic disorders.We have identified notoginsenoside Ftl(Ftl) from Panax notoginseng as an agonist of TGR5 in vitro.However,the pharmacological effects of Ftl on diet-induced obese(DIO) mice and the underlying mechanisms are still elusive.Here we show that Ftl(100 mg/100 diet) increased adipose lipolysis,promoted fat browning in inguinal adipose tissue and induced glucagon-like peptide-1(GLP-1) secretion in the ileum of wild type but not Tgr5^(-/-) obese mice.In addition,Ftl elevated serum free and taurineconjugated bile acids(BAs) by antagonizing Fxr transcriptional activities in the ileum to activate Tgr5 in the adipose tissues.The metabolic benefits of Ftl were abolished in Cyp27 al^(-/-) mice which have much lower BA levels.These results identify Ftl as a single compound with opposite activities on two key BA receptors to alleviate high fat diet-induced obesity and insulin resistance in mice.

Pro-angiogenic Activity of Notoginsenoside R1 in Human Umbilical Vein Endothelial Cells in vitro and in a Chemical-Induced Blood Vessel Loss Model of Zebrafish in vivo

Objective： This study aimed at investigating whether notoginsenoside R1 （R1）, a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells （HUVECs） and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish. Methods： The in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rgl and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit lI （XTI＇） assay. R1, Rgl and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigel- coated wells was performed to evaluate the pro-angiogenic actions of RI. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin （wort） or L-N （o -nitro- L-arginine methyl ester hydrochloride （L-NAME）, SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor （VEGF） receptor kinase inhibitor ]1 （VRI） for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels （ISVs） in zebrafish were assessed for the restorative effect of R1 on defective blood vessels. Results： R1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 （a VEGF-KDR/FIk-1 inhibitor）. Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase （PI3K）-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase （eNOS） inhibitor] or SH-6 （an Akt pathway inhibitor） significantly abrogated the R1 induced proliferation of HUVECs. In chemically- induced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs. Conclusion： R1, similar to Rgl and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/FIk-1 and PI3K- Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.